I have succeeded in extracting DNA from mosquitoes kept on silica or freezer, using appropriate kits. However, in the older samples and preserved on naphthalene, the extracted product is inadequate for DNA amplification and sequencing. Any suggestions for the optimization of an extraction protocol?
Hi, I used DNAzol modified protocol for the extraction of mosquitoes DNA from small samples (such as single spermtheca or individual ovaries samples) for qRT-PCR. It's a fast, easy, non-expensive and straightforward protocol. I don't know if naphtalene could affect DNA yield or quality. Maybe worth the try with a non important mosquito sample if you have one.
Thanks Paolo. I'll try to use DNAzol and later will comment here
You're welcome. Just few tips and suggestions. Use a small volume for the homogenization 20-80 ul depends on sample size to ensure a proper sample disgregation. Use a plastic pestle, press and gently rotate. Do not vortex the sample, this may result in DNA fragmentation. Then adjust the volume to 200-500ul washing the pestle with the freshly added DNAzol.
In the protocol, the manufacturer says that debris clarification step is optional and not necessary. I'd like to say that is absolutely recommended, just matter of 10 min centrifugation at 10000 x g on a benchtop centrifughe. Then uncap the vial and firmly invert the opened vials into a new tube. Keep the vial upside down and remove the remaining droplet with a p200 and discard the old vial. This help a lot with purity and quality.
For small sample, during precipitation, I'd also like to suggest you to add glycogen (as a DNA carrier for very small samples), this help also a lot. Remember to repeat the centrifugation after each 75% ethanol washing. It's not specified into the manufactures's protocol but washing and discarding without centrifugation will result in DNA loss.
Finally remember to let ethanol completely dry before resuspending the DNA if you need it for PCR.
If you have enough time you can also heat the resuspended DNA for 10 minutes at 70°C.
good luck!
Paolo. Thank you so much. The tips you sent are being useful. I'm modifying the extraction protocol to a mosquito leg single according to your tips. However, I'm one doubts: I have a glycogen solution with blue marker for precipitation reactions. I use to precipitate PCR products order to reading process of the sequence.
For now, I'll do the extraction using a kit with silica column (Qiagen). In that case, in which step I use glycogen? Considering that in the step last the DNA is washed passing through column.
I ask, add the glycogen along with the diluent, which will pass through the column carrying the DNA (if that is really possible, lol) and subsequently precipitates it, or make a precipitation reaction and purification after extraction?
I consider your answer. Thanks
Well I guess the only step you could upgrade with a single leg extraction on a silica column could be the crucial elution step. In fact the DNA yield is influenced only by salt concentration/pH of eluition solution and the volume of the eluition solution itself. You can't modify the composition of the eluition buffer (it is optimized) but you can play with the volume and temperature. By the way a single leg carry a very small amount of DNA and indeed you can't use a massive volume of eluition buffer (this will result in overdilution of sample). Glycogen in this case probably will not help for the purpose, it is excellent for in-solution purification but useless for solid phase purification. Anyway heating the eluition buffer at 60-65*C and reloading the eluate one more time on the column and pass it through twice usually increase the yield.
P.s. Is easy to swop glycerol with glycogen they sounds similar. Maybe your solution with blue marker (bromophenol blue maybe) could be a loading solution? In this case is useful for gel loading but less or nothing for nucleic acida precipitation.
Thanks Paolo. I will try to optimize the extraction protocol of a leg of old mosquitoes preserved in mothballs, with other modifications and considering your tips as well. I will report the results here.
Thank u so much ;)
Ps. The glycogen that I mentioned is below. Blue only serves to improve the viewing pellet.
GlycoBlue™ Coprecipitant (15 mg/mL)
http://www.thermofisher.com/order/catalog/product/AM9515?ICID=search-product
Cool product! For in-solution extraction and precipitation of DNA or RNA (i.e. For both DNAzol and Trizol protocols) 2-4 ul of your glycoblue will be absolutely perfect.
Good luck!
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